An NHEJ-independent Role for DNA-PKcs in ATR activation at DNA Double-Strand Breaks

ABSTRACT DNA double-strand breaks (DSBs) are threats to genome integrity, and to mitigate this risk cells activate the Ataxia Telangiectasia and Rad3-related (ATR) kinase, which halts cell cycle progression to allow time for repair. While ATR signalling during replication stress is well understood, how ATR is activated at DSBs remain unclear. Topoisomerase 2-Binding Protein 1 (TOPBP1) is a key activator of ATR, and activation is mediated by phosphorylation of TOPBP1 at Serine 1131 (S1131). Previous work showed that the Ataxia Telangiectasia Mutated (ATM) kinase phosphorylates TOPBP1 at S1131. ATM is primarily linked to the homologous recombination (HR)-based repair of DSBs, however the majority of cellular DSBs are repaired via the Non-Homologous End-Joining (NHEJ) repair pathway, raising the question of how (or if) ATR is activated in an ATM-independent manner. Here, using Xenopus egg extracts, we demonstrate that DNA-PKcs controls a pathway acting in parallel to ATM that promotes ATR signalling at DSBs. We show that, like ATM, DNA-PKcs phosphorylates TOPBP1 at S1131. DNA-PKcs is best known for orchestrating NHEJ, however we find that its roles in NHEJ and ATR signalling are separable. Our findings reveal an alternative pathway for ATR activation in which DNA-PKcs directly couples DSB recognition to ATR signalling. GRAPHICAL ABSTRACT